This is an application for a K01 Award for Dr. Baran A. Ersoy, an Instructor in Medicine at the Brigham and Women's Hospital (BWH) and Harvard Medical School (HMS). Dr. Ersoy is a young investigator in basic research on disorders of glucose and lipid metabolism. His application outlines a short-term plan to further his education by gaining experience in advanced molecular biology and immunology techniques, including mass spectrometry of proteomics and bone marrow transplantation. This plan will build upon his previous experience and provide him the time and training to establish new research skills, strategies and projects that will contribute to his success upon becoming an independent scientist. It is his long-term goal to become an establish independent investigator in an academic setting and study the mechanisms driving insulin resistance and promoting type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). To achieve these goals, Dr. Ersoy will be mentored by Dr. David E. Cohen, Professor of Medicine at HMS and Director of Hepatology at BWH. Dr. Ersoy has also assembled an advisory committee comprised of Dr. Steven P. Gygi, Professor of Cell Biology at HMS, who focuses on mass spectrometry of proteomics; Dr. Umut Ozcan, an endocrinologist with expertise in endoplasmic reticulum stress, inflammation and insulin signaling; and Dr. Morris F. White, an expert in insulin signaling who played a major role in the discovery insulin receptor substrate proteins. Dr. Ersoy's mentor and advisory committee members have established mentorship and training record, whose previous trainees hold independent investigator positions throughout the world. Dr. Ersoy will conduct his training at BWH and HMS, which will provide an intellectually rich research environment and has a long- standing commitment to training young scientists for independent academic research careers. Obesity-induced resistance to insulin action is the primary pathophysiological defect that predisposes to NAFLD and type 2 diabetes. Because current management options remain limited, identification of new regulatory mechanisms that govern the metabolic response to insulin should serve to identify novel opportunities for pharmacologic intervention. The objective of this research proposal is to elucidate a fundamental new mechanism for the regulation of glucose homeostasis. The rationale is that the identification of a novel mechanism that regulates hepatic insulin sensitivity could lead to the identification of new therapeutic targets for the treatment of NAFLD and type 2 diabetes. Guided by extensive preliminary data, the central hypothesis of this research plan is that phosphatidylcholine transfer protein (PC-TP) regulates glucose homeostasis by controlling the activity of insulin receptor substrate 2 (IRS2) both directly and also indirectly by modulating endoplasmic reticulum (ER) stress and inflammation. This will be tested in three specific aims: 1) Define the mechanisms by which PC-TP suppresses IRS2 activity; 2) Determine the role of ER stress in PC- TP-mediated regulation of insulin signaling; 3) Elucidate the role of immune response in PC-TP-mediated inhibition of insulin sensitivity. In Aim 1, a small molecule inhibitor of PC-TP (compound A1) and IRS2 deficient mice will be used to examine the role of IRS2 in the regulation of insulin signaling by PC-TP. The biological pathways in the control of IRS2 inhibition by PC-TP will be unveiled using mass spectroscopy of proteomics in Pctp+/+ and Pctp-/- mice as well as compound A1-treated hepatocytes harvested from these mice. In Aim 2, Pctp+/+ and Pctp-/- mice and hepatocytes harvested from these mice will be used to determine whether PC-TP modulates key ER stress pathways that regulate insulin signaling and IRS2 activity by immunoblot analyses. ER stress signaling will be activated by refeeding following 24 h starvation in mice, and by the chemical reagent tunicamycin in cell culture systems. Aim 3 will utilize peritoneal macrophages harvested from Pctp+/+ and Pctp-/- mice and bone marrow transplantation of Pctp-/- donor cells into Pctp+/+ recipient mice. Inflammation will be induced by high fat diet feeding in mice, and by lipopolysaccharide treatment in cell culture. Inflammatory signals will be measured by ELISA, immunoblot and qPCR analyses. It is anticipated that PC-TP will regulate insulin signaling by regulating IRS2 activity both directly and by modulating ER stress and inflammation. This is important because loss of IRS2 expression and increased ER stress and inflammation are associated with development of insulin resistance. These studies are expected to identify new therapeutic targets for the management of insulin resistance and related disorders. The data generated from this research proposal will form the basis for an R01 grant application at the end of the K01 award.